Question

After you performed the freeze-thaw cycle to lyse the bacteria, you pelleted the debris using a centrifuge. What two reasons did this pellet fluoresce green? (Yes, I know it contained GFP…. but why?)

Answer 1

Ans) Two main reasons for flourescence in pallets obtained from centrifugation of lysed cells are:

The pellet obtained after Centrifugation of dead(lysed) cells glowed green because the first reason is there is a presence unlysed cells in the pellet since the freeze-thaw method is not 100% efficient. This would cause some GFP protein within cells to be in the pellet instead of in the supernatant because the E.coli would still contain the GFP protein.

Second reason is that the pellet fluoresced green could be because the GFP(Green fluorescent protein) protein may be left in the cellular debris. Since all the cellular debris moves down to the pellet, some GFP would remain in the pellet as well.

• Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules especially Bacterias in living cells, and it has quickly became a primary tool for analysis of DNA and protein localization in prokaryotes.
• Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates.
• Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants.
• The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities.
• The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria.

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